Abstract Opioid based treatments are highly effective at relieving acute pain, but repeated or prolonged use of the drugs results in tolerance to their analgesic effects. Opioid receptor desensitization is an early step in the development of tolerance. Therefore, understanding the mechanisms that govern receptor desensitization is a critical step in designing approaches to reduce desensitization and possibly prevent analgesic tolerance. The work proposed will build on the observation that acute opioid receptor desensitization occurs selectively at somato-dendritic mu opioid receptors (MORs), whereas MORs located on presynaptic terminals of neurons maintain signaling during opioid treatment. Our recent studies provided strong evidence that presynaptic resistance to desensitization is not specific to the MOR but is a property of many inhibitory GPCRs. It is currently unclear whether a property of the receptor or the presynaptic compartment confers resistance to desensitization. The experiments in this proposal will 1) determine how effector coupling and phosphorylation influence a receptor's ability to resist desensitization, and 2) determine whether a receptor's diffusion state is related to its ability to desensitize. Hypopthalamic proopiomelanocortin (POMC) neurons receive inputs that are regulated by MORs and GABAB receptors (GABABRs). MORs presynaptic to POMC neurons are completely resistant to acute desensitization, but approximately ~25% of POMC neurons receive inputs wherein GABABR mediated inhibition of GABA release robustly desensitizes. Using brain slice electrophysiology and pharmacological tools, it will be determined whether this differential desensitization between MORs and GABABRs can be attributed to a particular effector pathway, or if desensitization occurs at the level of the receptor. To determine the role that the diffusion state of the receptor plays in receptor desensitization, single-particle tracking of fluorescently tagged somato-dendritic MORs in cultured neurons will be utilized. It is hypothesized that agonist treatment will alter the mobility of MORs as they undergo desensitization. Differences in diffusion state may be a key factor in differential desensitization of pre- and postsynaptic MORs. The data generated in Aim 2 will provide a platform for future studies investigating presynaptic receptors specifically. Altogether, the data will provide valuable information about the mechanisms underlying resistance to desensitization by presynaptic MORs. Understanding how resistance to desensitization occurs will provide information that may be used in the rational design of new opioid agonists that are both highly efficacious and produce limited tolerance. In performing the proposed experiments the applicant will gain valuable training that will provide a solid platform for an independent scientific career.